Bio-active proteins produced by cells have been widely utilized in industry as materials for medicines, diagnosis, biosensors, bio-reactors and so on. The progress of gene technology has facilitated the discovery of these proteins and enabled us to produce a large amount of the proteins. The fundamental technology is a cDNA cloning technique.
Information on the amino acid sequence of a protein is encoded by mRNA. If the mRNA is converted to a corresponding DNA, i.e., a complementary DNA (cDNA), the cDNA can be used for determining the primary structure of the protein and producing a large amount of the protein. Therefore, various cDNA cloning techniques have been developed which include isolating mRNA from cells, synthesizing cDNAs from the mRNA, and isolating a cDNA encoding a target protein.
The most important requisite for cDNA cloning is to synthesize a cDNA containing an entire protein coding region encoded by an intact mRNA. The intact mRNA contains a characteristic structure at the 5' terminal, so called a cap. The cDNA containing an entire sequence involving from the nucleotide at which the cap is added is called a "full-length cDNA". If a full-length cDNA is synthesized, one can know the whole information on the primary structure of a protein and also use it for mass production of the protein encoded by the cDNA. According to conventional terminology, a "full-length cDNA" has been used as a cDNA containing an entire coding region of the protein even if the cDNA contains no cap site sequence. In this invention, however, a "full-length cDNA" is defined only as a cDNA containing a nucleotide sequence starting from the cap site.
The Gubler-Hoffman method [Gene 25:263-269(1983)], which is most widely used for cDNA synthesis, could not give a full-length cDNA, because deletion at the terminals of cDNA occurs. On the other hand, the Okayama-Berg method [Mol.Cell.Biol. 2:161-170(1982)] is known to give a full-length cDNA at high efficiency. The characteristic feature of this method is to add a dC tail to the 3' end of the first strand cDNA synthesized from mRNA. This method, however, does not always give a full-length cDNA, because dC tailing also occurs on truncated cDNA generated by unexpected termination of cDNA synthesis. The truncated cDNAs originated from the degraded mRNAs are also synthesized.